Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins

Abstract

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored.IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.

Keywords: Mycoplasma; exosecretion; posttranslational cleavage; proteases.

FIG 1

Casein digestion on an agar plate (modified Hayflick’s medium) supplemented with 0.4% (wt/vol) milk. An area of casein digestion was observed for strains producing extracellular caseinolytic proteases. The strongest activity was observed for M. mycoides subsp. capri strain 95010 (panel 1), while a digested zone was evidenced for M. capricolum subsp. capripneumoniae Abomsa, although no conspicuous growth was observed for that fastidious strain (panel 2). The activity observed for M. capricolum subsp. capricolum Ck strain (panel 3) was completely abolished in the Ck-mut strain (panel 4) although its culture was clearly visible on the agar surface. Lower activity was evidenced for M. mycoides subsp. mycoides Rita (panel 5), and no activity was recorded for the mutated Rita-mut strain (panel 6). Slight activity was recorded for M. bovirhinis MV5 (panel 7).

FIG 2

Detection of caseinolytic activity by zymography. Supernatant from mycoplasmas was incubated in supplemented Opti-MEM. (A) Casein zymogram. (B) SDS-PAGE. Lanes L, Ladder PageRuler (top to bottom, 180, 130, 100, 70, 55, 40, 35, and 25 kDa); lane 1, M. bovirhinis MV5; lanes 2, M. mycoides subsp. capri 95010; lanes 3, M. capricolum subsp. capripneumoniae Abomsa; lane, 4, M. capricolum subsp. capricolum 94157; lanes 5, M. capricolum subsp. capricolum Ck-mut; lanes 6, M. capricolum subsp. capricolum Ck; lane 7, M. mycoides subsp. mycoides Rita; lane 8, M. mycoides subsp. mycoides Rita-mut. The discolored bands, showing caseinolytic activity, were cut and then analyzed by tandem mass spectrometry. The zymography picture (panel A) has been spliced to remove the lanes with samples which did not yield any digested bands (M. arginini, M. bovis, M. ovipneumoniae, and M. mycoides subsp. mycoides).

FIG 3

Identification of peptides detected by tandem mass spectrometry for the predicted S41 protease (CBW53985.1, MLC_2570) of M. mycoides subsp. capri strain 95010. The N-terminal and C-terminal transmembrane regions are boxed. The predicted S41 superfamily domain is represented in bold with a boxed serine active site. The specific peptides detected by tandem mass spectrometry in the concentrated supernatant are underlined.